Binding of the transcription factor Ets-1 to DNA is allosterically regulated by a serine-rich region (SRR) that modulates the dynamic character of the adjacent structured DNA-binding ETS domain and its flanking autoinhibitory elements. Multi-site phosphorylation of the flexible SRR in response to Ca(2+) signaling mediates variable regulation of Ets-1 DNA-binding affinity. In this study, we further investigated the mechanism of this regulation. First, thermal and urea denaturation experiments demonstrated that phosphorylation of the predominantly unstructured SRR imparts enhanced thermodynamic stability on the well-folded ETS domain and its inhibitory module. We next identified a minimal fragment (residues 279-440) that exhibits both enhanced autoinhibition of Ets-1 DNA-binding and allosteric reinforcement by phosphorylation. To test for intramolecular interactions between the SRR and the rest of the fragment that were not detectable by (1)H-(1)H NOE measurements, paramagnetic relaxation enhancements were performed using Cu(2+) bound to the N-terminal ATCUN motif. Increased relaxation detected for specific amide and methyl groups revealed a preferential interaction surface for the flexible SRR extending from the inhibitory module to the DNA-binding interface. Phosphorylation enhanced the localization of the SRR to this surface. We therefore hypothesize that the positioning of the SRR at the DNA-binding interface and its role in shifting Ets-1 to an inhibited conformation are linked. In particular, transient interactions dampen the conformational flexibility of the ETS domain and inhibitory module required for high-affinity binding, as well as possibly occlude the DNA interaction site. Surprisingly, the phosphorylation-dependent effects were relatively insensitive to changes in ionic strength, suggesting that electrostatic forces are not the dominant mechanism for mediating these interactions. The results of this study highlight the role of flexibility and transient binding in the variable regulation of Ets-1 activity.